Structural Basis of Opioid Receptor Function

描述（由申请人提供）：本提案的目的是确定阿片受体药理学和功能的结构基础。阿片受体是治疗疼痛的主要和最有效的靶点。然而，在欧洲和北美，作用于这些受体的阿片类药物的使用是过量死亡的主要原因。阿片类药物（鸦片、海洛因）和已批准的治疗药物（吗啡和可待因）的利与副作用均由μ阿片受体（μ-OR）的激活介导，μ-OR是G蛋白偶联受体的成员 （GPCR）超家族。为了理解μ-OR功能的结构基础，我们获得了该受体的非活性状态的第一个高分辨率晶体结构，该受体沿着有一个密切相关的家族成员δ-阿片受体（δ-OR）。这些结构为特定拮抗剂药物的OR偏好提供了新的见解，但没有解决有关OR激活机制的重要问题，特别是阿片类药物的疗效。因此，我们建议使用生物化学和生物物理方法的组合来表征阿片受体激活的结构基础。具体目标包括：目标1。确定OR的活性态结构μ-OR和δ-OR的非活性态结构提供了对吗啡喃拮抗剂结合模式的第一个结构见解。这个目标的目的是获得阿片受体激活G蛋白Gi的过程中的结构见解。我们将首先专注于使用晶体学和单粒子电子显微镜来获得μ-OR-Gi和μ-OR-Gi复合物的三维结构。我们还将开发一个 一组骆驼抗体片段（纳米抗体），稳定了晶体学的配体特异性构象状态。这些将用于确定阿片受体激动剂不同功能特性的结构基础。目标2. OR结构和激活的构象动力学。OR活化和更一般地GPCR活化涉及配体结合和G蛋白偶联结构域之间的复杂变构偶联，其知之甚少。晶体结构提供了这个动态过程的有限数量的静态快照。因此，我们建议开发和应用生物物理的方法来表征结构的可塑性和动态特性的OR，并确定这是如何转化为信号的复杂性和配体的功效。非活性态晶体结构将构成设计和解释生物化学和生物物理研究的重要起点，其中一些研究包括荧光、EPR和NMR光谱。这些研究将为阿片类配体的功效以及小分子和肽配体之间的差异提供新的见解。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the structural basis for opioid receptor pharmacology and function. Opioid receptors constitute the major and the most effective target for the treatment of pain. The use of opioid drugs acting at these receptors is however a leading cause of death by overdose in Europe and North America. Both beneficial and adverse effects of illicit opioid drugs (opium, heroin) as well as approved therapeutics (morphine and codeine) are mediated by the activation of mu opioid receptor (μ-OR), a member of the G protein-coupled receptors (GPCRs) superfamily. To understand the structural basis for μ-OR function, we obtained the first high-resolution crystal structure of an inactive state of this receptor along with a closely relate family member the delta-opioid receptor (δ-OR). These structures provide new insights into OR preference for specific antagonist drugs but do not address important questions regarding OR activation mechanisms and, in particular, opioid drug efficacy. We therefore propose to characterize the structural basis of opioid receptor activation using a combination of biochemical and biophysical approaches. Specific Aims include: Aim 1. Determine active state structures of ORs The inactive state structures of the μ-OR and δ-OR provided the first structural insights into the binding mode of morphinan antagonists. The goal of this aim is to obtain structural insights into the process of opioid receptor activation of the G protein Gi. We will initially focus on the use of crystallography and single particle electron microscopy to obtain three-dimensional structures of the μ-OR-Gi and ∂-OR-Gi complexes. We will also develop a panel of camelid antibody fragments (nanobodies) that stabilized ligand-specific conformational states for crystallography. These will be used to determine the structural basis for the different functional properties of opioid receptor agonists. Aim 2. Conformational dynamics of OR structure and activation. OR activation and more generally GPCR activation involves a complex allosteric coupling between ligand binding and G protein coupling domains that is poorly understood. Crystal structures offer a limited number of static snapshots of this dynamic process. We therefore propose to develop and apply biophysical approaches to characterize the structural plasticity and dynamic properties of ORs and to determine how this is translated into signaling complexity and ligand efficacy. Inactive state crystal structures will constitute an important starting point for designing and interpreting biochemical and biophysical studies, some of which include fluorescence, EPR and NMR spectroscopy. These studies will provide new insights into opioid ligand efficacy and the differences between small molecule and peptide ligands.